5 h of batch fermentation. For the preliminary fed-batch studies, two predetermined feeding profiles, namely exponential and constant feeding were preferred. For each feeding profile, three feeding rates were evaluated: 1, 3 and 6 g

glycerol/L/h for constant feeds and 0.1, 0.2 and 0.3 h−1 for exponential feeds. To achieve the desired rates (1, 3 and 6 g glycerol/L/h), several feed mediums with different glycerol concentrations were prepared. For these assays, the three feeding rates were tested as duplicates (A and B), without induction, so that the growth profiles could be established (Fig. 3). In these fed-batch experiences, glycerol was measured as mentioned in Section 2.2.4 until the end of the feeding see more process. The growth curves for these Apitolisib profiles (Fig. 3) show a maximum OD of about 50 which, as expected, is considerably higher than those obtained in the batch experiments. For the 1 g/L/h constant feeding profile, glycerol concentration was kept close to zero until the end of the fed-batch process, meaning that these cultures were able to consume all of the glycerol provided by the feeding

solution. For the 3 g/L/h constant feeding profile, glycerol concentration reached close to zero values only after about 10 h of fed-batch, meaning that limiting concentrations are not reached during most of the fed-batch process. However, the maximum OD reached (52) was very similar to that of the 1 g/L/h feeding profile. Finally, for the 6 g/L/h feeding profile, glycerol concentrations either increased throughout the experiment (replicate A) or were kept constant at relatively low levels (replicate B). Since glycerol concentrations during the fed-batch phase of the feeding profiles evaluated were very different (from almost 0 g/L to as high as 30 g/L), cytometry assays were used to see if the feeding profile of 1 g/L/h was, in

fact, the best choice among the three constant feeding profiles tested. In order to assess cell physiology during the fed-batch experiments, flow cytometry assays were carried out using a PI/BOX dual staining. Dead cells will be stained with both BOX and PI, cells with depolarized membrane will be stained only with BOX and viable cells will not be stained. The results (not shown), indicate that as fermentation time increases, the percentage of dead cells (stained with PI and BOX) also increases. This effect is heightened at Clomifene higher feeding rates, possibly because of the higher glycerol concentrations, which can hamper E. coli growth. In fact, at the end of the fermentation, the average percentages of viable cells were 79.43, 65.84 and 75.61% for 1, 3 and 6 g/L/h, respectively. The three chosen specific growth rates for exponential feeding profiles were 0.1, 0.2 and 0.3 h−1 with feed medium addition speed being calculated according to an equation previously described [14]. For this set of experiments, the three specific growth rates were also performed in duplicates (A and B) without induction (Fig. 4).